WO2007115713A1

WO2007115713A1 - Treatment of multiple sclerosis and/or rheumatoid arthritis

Info

Publication number: WO2007115713A1
Application number: PCT/EP2007/002929
Authority: WO
Inventors: Marco Prinz; Wolfgang BRÜCK; Alexander Mildner; Matthias Mack
Original assignee: Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts; Klinikum Der Universität Regensburg - Anstalt Des Öffentlichen Rechts -
Priority date: 2006-04-03
Filing date: 2007-04-02
Publication date: 2007-10-18
Also published as: AU2007236269B2; US20090311273A1; US9068002B2; EP2004692A1; EP2481757A1; CA2648330C; CN101466735A; JP2009532399A; DE102006015341A1; CA2648330A1; AU2007236269A1; EP2004692B1

Abstract

The invention relates to the use of an antibody which can specifically bind to chemokine receptor CCR2 for producing a medicament utilized for the treatment of multiple sclerosis and/or rheumatoid arthritis in a subject that is preferably a primate or a human. In another embodiment, the invention relates to the use of an antibody which can specifically bind to chemokine receptor CCR2 for producing a medicament that is utilized for depleting monocytes in subjects suffering from multiple sclerosis and/or rheumatoid arthritis. The invention further relates to corresponding in vitro methods and therapeutic methods. An antibody which binds to CD 14, for example, can be used in addition to the antibody that can bind to CCR2.

Description

Treatment of multiple sclerosis and / or rheumatoid arthritis

The present invention relates to the treatment of multiple sclerosis and / or rheumatoid arthritis using antibodies.

Multiple sclerosis is a central nervous system disorder in which the medullary sheaths of the nerve fibers are destroyed, i. the nerve fibers are demyelinated. Cause and pathophysiology are poorly understood. Presumably it is an exogenous and endogenously modulated immunological process. The symptoms of multiple sclerosis are relatively non-specific and include, for example, spastic paralysis and other motor and sensory deficits.

A satisfactory therapy for multiple sclerosis is not yet possible. For example, therapy has heretofore been accomplished by high dose administration of steroids, as well as by administration of azathrioprine, glatiramer acetate, methotrexate, mitoxantrone, or by immunomodulatory treatment, e.g. with interferons. However, this only leads to a reduction in the frequency and intensity of the disease relapses. Otherwise, the treatment is based on alleviating the symptoms.

Schneider et al. mention that depletion of cells expressing the chemokine receptor CCR2 may represent a potential strategy for the treatment of inflammatory diseases (Schneider, MA, Bruehl, H. Wechselberger, A., et al. (2005), In Vitro and In Vivo properties of a dimeric bispecific single-chain antibody IgG fusion protein for depletion of CCR2 ⁺ target cells in mice, European Journal of Immunology, 35, pp. 987-995). The authors describe the lysis of CCR2-positive cells in mice using a bispecific antibody, which is directed, inter alia, against CCR2. However, no therapeutic effect is shown.

Rheumatoid arthritis is a chronic inflammatory systemic disease that predominantly affects the joints and leads to deformities and restricted mobility.

Symptoms include, for example, swelling, pain and restricted mobility. The The cause of rheumatoid arthritis is still unclear, including immunopathogenesis, infectious causes and genetic factors.

The therapy of rheumatoid arthritis is typically with so-called anti-inflammatory drugs, i. e.g. with DMARDs (disease modifying antirheumatic drugs), which include, for example, methotrexate and leflunomide. Additionally, steroids can be used for

Use come. In addition, biologics or immunosuppressive agents, e.g. TNF-alpha

Blockers (e.g., TNF-alpha or Ethernacept monoclonal antibodies) or IL-1

Receptor antagonists (e.g., Anakinra). Nonsteroidal anti-inflammatory drugs (NSAIDs) are mainly used for pain therapy. Nevertheless, it succeeds in many

Not to treat the disease satisfactorily and joint damage that persists

Immobilization are going to be avoided.

Brühl et al. describe the administration of monoclonal antibodies (MC-21) directed against the chemokine receptor CCR2 in the mouse model of collagen-induced arthritis (Bruhl H, Cihak J, Schneider MA, Plachy J, Rupp T, Wenzel I, Shakarami M, Spleen S, Ellwang JW, Stangassinger M, Schlondorff D, Mack M. (2004) Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2 + T cells. J Immunol., Vol. 172 (2), Pp. 890-898). They describe that antibody relief was only achieved if the blockade of CCR2 occurred during the initiation phase of the disease (ie immediately after immunization with collagen and before the onset of arthritis), but not during the progression phase. During the progression phase, blockade of CCR2 even aggravated the arthritis and immune response to collagen. It can be concluded that the antibodies only influenced the artificial production of the disease in the model system, ie the immunization of the animal with collagen. Treatment of the disease itself, however, does not seem possible in this way.

Brodmerkel et al. (2005) describe a small molecule antagonist, INCB3344, of the mouse CCR2 receptor (Brodmerkel, CM., Huber, R. Covington, M., Diamond, S., Hall, L., et al. (2005) Discovery and Pharmacological Characterization of a Novel Rodent Active CCR2 Antagonist, INCB3344). The molecule was studied in the mouse model of experimental autoimmune encephalitis (EAE), a model of multiple sclerosis. However, administration already started at a time point (0 days or 7 days after immunization) when only a few animals showed clinical signs of the disease. Brodmerkel et al. (2005) further describe the administration of INCB3344 in rats in the model of adjuvant-induced arthritis. The administration started again at a point in time (9 days after immunization) at which the rats showed little or no clinical signs of the disease.

In conclusion, there is currently no satisfactory therapy for multiple sclerosis and / or rheumatoid arthritis.

Against this background, the object of the present invention is to provide a new treatment option for multiple sclerosis and / or rheumatoid arthritis.

This object is achieved by a medicament containing an antibody which can specifically bind to the chemokine receptor CCR2, for the treatment of multiple sclerosis and / or rheumatoid arthritis in a subject or the use of such an antibody for the manufacture of a medicament for the treatment of multiple sclerosis and / or rheumatoid arthritis in a subject.

Surprisingly, it was found in the context of the present invention that the administration of antibodies directed against CCR2, in contrast to the previous literature (see in particular Brühl et al., 2004, cited above), can bring about a clinical improvement of multiple sclerosis and / or rheumatoid arthritis In fact, it has been found that by administering antibodies directed against CCR2 a clinical improvement in rheumatoid arthritis can be achieved, preferably at a dosage lower than the dose chosen by Brühl et al. (2004). that a clinical improvement of multiple sclerosis can be effected.

Furthermore, it has surprisingly been found that treatment is possible not only during the induction of the disease in an animal model, but also when there are already clear clinical symptoms of the disease. Among other things, this allows a relatively late therapeutic intervention, as required in clinical practice in humans.

The chemokine receptor CCR2 is known to the person skilled in the art (see, for example, entry no. 601267 in the database Online Mendelian Inheritance in Man (see OMIM on the website of the National Institutes of Health, USA: http://www.ncbi.nlm.nih.gov/ )). It is a CC type chemokine receptor present in two different splice variants in humans, CCR2A and CCR2B (Wong, L.-M., Myers, SJ, Tsou, CL., Et al. (1997)). Organization and Differential Expression of the Human Monosite Chemoattractant Protein 1 receptor gene: Evidence for the role of the carboxyl terminal tail in Receptor Trafficking. Biol. Chem., Vol. 272, pp. 1038-1045). The molecule is also known as human monocyte chemoattractant protein-1 receptor gene. It is a 7-transmembrane domain G protein-coupled receptor. The corresponding orthologues of CCR2 from several other species are also known and can be easily determined by the skilled person, for example by means of sequence searches starting from the human CCR2.

The term antibody is known to those skilled in the art. According to the present invention, the term antibody is to be understood in a broad sense and includes, inter alia, polyclonal, monoclonal and recombinantly produced antibodies, as well as fragments thereof, such as Fv, Fab and F (ab) ₂ fragments, which are single-stranded (single -chain). Preferably, the antibody is of the isotype IgG. Preferably, the antibody is of an isotype capable of inducing effector mechanisms such as ADCC (antibody dependent cellular toxicity) or complement activation. This would be the case, for example, with human IgG1 or mouse IgG2a. Preferably, the antibody is such that it is not repelled by immune system of the subject as possible or only to a limited extent. This can be achieved, for example, in that the regions not required for the recognition of the antigen (in this case the CCR2 receptor) (eg the Fc regions) are formed from antibody sequences of the species of the subject. For example, those skilled in the art so-called humanized antibodies are known, which are particularly suitable for use in humans. The term "antibody" for the purposes of the present invention also includes other modified antibodies, for example bispecific antibodies (see eg Schneider et al. (2005), cited above), diabodies and so-called binders or aptamers, which are synthesized, for example, with the aid of a peptide Backbone or a Nukleinsäurerückgrates, such as an RNA can be formed. Preferably, for the purposes of the present invention, the antibodies have a molecular weight of less than 600 kDa, more preferably less than 300 kDa, even more preferably less than 200 kDa, and most preferably about 150 kDa. The preparation of suitable polyclonal, monoclonal and recombinant antibodies, including the preparation of binders and aptamers, is known to the person skilled in the art (see, for example, Jörg Knäblein (ed.), Modern Biopharmaceuticals, Vol. 2, p. 635), see also exemplary embodiment. For example, immunization may be by injection of cells expressing the CCR2 receptor. This has the advantage that the receptor is used as an antigen in its native fold. Furthermore, immunization with peptide fragments of the antigen is possible. Preferably, corresponding peptides are extracted from the extracellular domains of CCR2, especially from the first two loops (Loops) selected. Identification of suitable antibodies (including binders and aptamers) is also possible by screening libraries of appropriate molecules or cells expressing these molecules, eg, by phage display. After identification, the antibodies (including binders and aptamers) can be prepared by methods known to those skilled in the art.

Preferably, the antibodies of the invention are coupled to domains that enhance the depleting effect of the antibodies. Such domains are known to those skilled in the art. An example of this is an Fc domain that induces ADCC or complement activation. Another possibility is the coupling of the antibody with a toxin. The antibody may also be a bispecific antibody, e.g. the bispecific variant of MC-21 described in Schneider et al. (2005) and recognizes as the second antigen the mouse CD3 epsilon (Schneider et al., 2005, cited above).

The antibody according to the present invention can bind to the chemokine receptor CCR2. Preferably, the antibody binds to Loopl and Loop2 of the extracellular CCR2 domains. Preferably, the antibody binds with a dissociation constant Kd of 10 ⁷ M or greater, more preferably 10 ⁸ M or greater, even more preferably 10 ⁹ M or greater, most preferably 10 ¹¹ M or greater. Preferably, the binding is specific. For the purposes of the present invention, specific binding means that the antibody binds to CCR2 under physiological conditions (ie for example in physiological saline solution, in cell culture or in vivo, preferably in the blood or tissue of the relevant subject) with at least 10-fold affinity, preferably 20-fold affinity, more preferably 50-fold affinity, most preferably 100-fold affinity for CCR2 compared to other proteins, especially when compared to similar proteins (eg compared to CCR5, CCR1, CCR3, or other chemokine receptors and G protein-coupled receptors with 7 transmembrane domains, preferably in comparison to CCR5). However, binding to other proteins may be tolerable unless it interferes with the therapeutic effect of the antibody. However, cross-reactivity with orthologous CCR2 receptors from other species is possible and may even be beneficial to allow use of the antibody in multiple species. Such cross-reactivity is not uncommon and it is known to those skilled in the art how to determine cross-reactivity. Further details can also be found in the exemplary embodiment.

Preferably, the antibody to be used according to the invention can inhibit the chemokine receptor CCR2. Whether an antibody inhibits the chemokine receptor CCR2 is easy to determine. For example, such an antibody reduces the MCP-I and MCP-3-induced chemotaxis of cells that chemotactically react with the aid of CCR2, eg, monocytes. Examples of corresponding cells from mouse or human are Monomac® cells or human monocytes from peripheral blood (PBMC). A corresponding assay is described in the exemplary embodiment.

Furthermore, the inhibition of the chemokine receptor CCR2 can be determined by the fact that an inhibition of the binding of MCP-I and the MCP-I-induced calcium influx occurs. A corresponding assay is described in the exemplary embodiment.

Examples of antibodies that may be suitable or a source of migrant antibody are the antibodies selected from the group consisting of DOC-I, DOC-2, DOC-3 and MC-21. The preparation of the antibodies DOC-I, DOC-2, DOC-3 and their properties are described in the embodiment.

The medicament according to the invention contains the antibody. In addition, the medicament may receive any type of excipients that the skilled artisan deems appropriate. Such adjuvants may be, for example, carriers, e.g. Starch, lactose, fats, stearic acid, alcohol, physiological saline solutions or other additives. In particular, excipients may be considered which stabilize and preserve antibodies.

The administration of the medicament may be carried out by any known method with which the antibody contained in the drug is delivered to the target cells, i. in particular monocytes, in vitro or in vivo can be introduced. For example, the drug may be administered by injection, e.g. intravenously (i.v.), subcutaneously (s.c.) or intraperitoneally (i.p.) in the form of solutions or infusions. But other forms of administration such. in microencapsulated form or in the form of implants are conceivable. Preferably, the administration of the medicament is such that the antibody can enter the bloodstream or the corresponding target area. Also conceivable is administration directly into the target area, for example in multiple sclerosis into the central nervous system, e.g. the cerebrospinal fluid, or in rheumatoid arthritis in the affected joints.

The diseases of multiple sclerosis and rheumatoid arthritis are known in the art and have already been described in the introduction.

The term of treatment is also known to the person skilled in the art. For the purposes of the present invention, the treatment is any kind of Intervention that causes a clinical improvement in multiple sclerosis and / or rheumatoid arthritis in a subject. A clinical improvement can be determined, for example, in multiple sclerosis, for example by measuring the decrease in neurological deficits, such as paralysis. In rheumatoid arthritis, a clinical improvement, for example, by decreasing the symptoms, such as the swelling, the inflammation of the pain can be determined. Preferably, the improvement manifests itself in a reduction in clinical symptoms, for example, an improvement in the EAE score according to the exemplary embodiment by at least 0.25 units, preferably at least 0.5 units, more preferably at least 0.75 units, most preferably at least 1 , 0 units equals. Preferably, the improvement manifests itself in a reduction of the clinical symptoms, for example, an improvement of the arthritis score according to the embodiment by at least 0.25 units, preferably at least 0.5 units, more preferably at least 0.75 units, more preferably at least 1, 0 units, most preferably at least 1.25 units. Many parameters are also known for the assessment of clinical improvement in humans, for example the EDSS score for multiple sclerosis (Expanded Disability Status Scale, see Kurtzke, JF (1983).) Rating neurologic impairment in multiple sclerosis: an expanded disablility status scale (cf. EDSS) Neurology, Vol. 33 (11), pp. 1444-1452)). Thus, the clinical improvement may be manifested in a reduction in symptoms, for example, an improvement according to the EDSS score of at least 0.5, preferably at least 1.0, more preferably at least 1.5 units, more preferably at least 2.0 units , most preferably at least 2.5 units. For the assessment of clinical improvement in humans, the ACR (American College of Rheumatology) score for rheumatoid arthritis is also known.

Preferably, the treatment relates to a subject suffering from multiple sclerosis and / or rheumatoid arthritis. In particular, the treatment relates to multiple sclerosis and / or rheumatoid arthritis during the disease, especially treatment in the presence of clinical signs of the disease. For example, clinical signs of the disease correspond to an EAE score (see embodiment) of 1.0 or greater, more preferably 1.5 or greater, even more preferably 2.0 or greater, even more preferably 2.5 or greater, more preferably 3.0 or greater , Clinical signs of the disease further correspond, for example, to an arthritis score (see embodiment) of 1.0 or greater, more preferably 2 or greater, even more preferably 3 or greater, even more preferably 4 per paw. Many parameters are also known for the assessment of clinical signs in humans, for example the EDSS score (Expanded Disability Status Scale, see Kurtzke, JF (1983).) Rating neurologic impairment in multiple sclerosis: an expanded disablility status scale (EDSS). Neurology, Vol. 33 (11), pp. 1444-1452)) and the ACR (American College of Rheumatology) score for rheumatoid arthritis.

Further, the treatment may particularly relate to therapy-refractory multiple sclerosis and / or rheumatoid arthritis, i. a form of the respective disease in which a clinical improvement could not be achieved with the previously known agents. In a significant number of subjects it comes to a course in which with previous means no clinical improvement is possible. Furthermore, the treatment may concern a subject with the particular disease, in whom with previous treatment methods unwanted side effects in unacceptable

Dimensions occur. Furthermore, the treatment may affect the respective disease in the advanced stage.

The subject in the sense of the present invention is a vertebrate, preferably a mammal. The mammal may be, for example, a rodent (e.g., mouse, rat, or rabbit), a pig, a dog, a cat, or primate. Preferably, the mammal is a primate (for example, a macaque or white-jacketed monkey or a human). Particularly preferably, the subject is a human.

For administration of the drug, an effective dose is usually determined. The term of the effective dose and the determination of the effective dose is known to those skilled in the art, and he can also rely on the information provided here to determine the effective dose. Effective is understood to be a dose at which clinical improvement of the treated disease occurs. In particular, for the purposes of the present invention, an effective dose is understood to be a dose of the medicament which brings about a reduction in the symptoms of multiple sclerosis and / or rheumatoid arthritis in a subject. An effective dose is, for example, a dose chosen to deplete at least 20%, preferably at least 30%, more preferably at least 40%, most preferably at least 50% of the CCR2-expressing monocytes in the peripheral blood. By depletion of the monocytes is meant a destruction of the cells.

Preferably, the effective dose of the drug is selected to be the lowest dose that will ensure satisfactory treatment success of multiple sclerosis and / or rheumatoid arthritis. A particularly suitable effective dose can be determined by increasing the dose in test series until the desired ratio of effect and undesired side effect is reached. In the context of the present invention this becomes be achieved, for example, if the treated disease is no longer clinically improved (possibly even enhanced) with further increase in the dose and / or if unwanted side effects in relation to the therapeutic effect are no longer acceptable.

For example, a dose which does not exceed that dose necessary to achieve a depletion of 99%, preferably 98%, more preferably 95%, more preferably 90%, more preferably 80%, more preferably 70%, even more preferably 50% of CCR2, should be considered to reach expressing monocytes in peripheral blood. Within the scope of the invention, it has been possible to show that a dose which, although it causes an extensive depletion of the monocytes, but is also not significantly higher, produces a remarkable clinical improvement and not an enhancement of the disease. In particular, this relates to rheumatoid arthritis.

Alternatively, the dose may be oriented on whether it causes release of interleukin 6 (IL-6) and / or interleukin-4 (IL-4). In particular, this orientation of the dose may be due to a release of IL-6. Thus, the dose may preferably be chosen to be low enough that it does not cause excessive release of IL-6 and / or IL-4. For example, the dose may be chosen to be no more than a 20-fold increase, preferably not more than 10-fold increase, more preferably no more than 5-fold increase, most preferably no more than 2-fold increase in the level of IL-6 and / or IL-4 in the blood plasma. In this context, the increase refers to the normal level of IL-6 and / or IL-4 in the blood plasma of the subject. As a "normal" level of IL-6 and / or IL-4, it is preferable in this context to refer to the level that the subject has before the administration of the drug. It is known to those skilled in the art when it may view release of IL-6 and / or IL-4 as caused by the administration of the medicament. For example, an increase in the level of IL-6 and / or IL-4 may be considered two to six hours after intraperitoneal injection of the antibody than caused by the drug.

In addition, as a suitable dose, consider a dose whose lower limit is optionally 0.01; 0.02; 0.03; 0.05; 0.07; 1.0; 1.5; 2.0; 2.5; or 3.0 mg of the antibody per kg body weight of the subject per day and the upper limit thereof optionally 1.0; 1.5; 2.0; 2.5; 3.0; 4.0; 5.0; 7.0; 10; 12; 14; 17; or 20 mg of the antibody per kg body weight of the subject per day, which limits can be arbitrarily combined, but selected to include a positive range of the boundaries. Examples of such areas are 0.01-20 or 0.01-2 or 2.5-20 mg of the antibody per kg body weight of the subject per day.

Furthermore, the present invention relates to a medicament containing an antibody capable of binding to the chemokine receptor CCR2 in multiple sclerosis and / or rheumatoid arthritis in a subject or the use of such an antibody for producing a medicament for depleting monocytes in multiple sclerosis and / or or rheumatoid arthritis in a subject.

Further, the present invention relates to a medicament containing an antibody capable of depleting monocytes of multiple sclerosis and / or rheumatoid arthritis in a subject or the use of such an antibody for the manufacture of a medicament for the treatment of multiple sclerosis and / or rheumatoid arthritis in a subject Subject.

All of the preferred embodiments and variants of the invention and definitions shown in this description naturally also relate to the two abovementioned medicaments and uses in a corresponding manner.

Within the scope of the present invention, it has been found that the administration of an antibody which can inhibit the chemokine receptor CCR2 can cause a depletion of CCR2-expressing monocytes.

It is assumed by the inventors that the treatment of multiple sclerosis and / or rheumatoid arthritis is preferably carried out via a depletion of CCR2-expressing inflammatory monocytes.

The depletion of monocytes in the context of the present invention means the destruction of the corresponding monocytes.

The mechanism of depletion may be influenced by the design of the antibody. Preferably, the depletion is by a cell lysis based on ADCC (antibody-dependent cellular toxicity) and / or complement activation. In the case of complement activation, however, care should be taken that there is no increased inflammatory reaction, for example via the complement molecules C3a and C5a. The depletion based on ADCC can be mediated, for example, via CD 16/32 or CD64. ADCC allows monocytes to deplete each other. A depletion with the aid of the antibodies according to the invention can be achieved in the most diverse species. For example, the monocyte populations of mouse and human are similar. For example, it is known that in the mouse a dichotomy of the blood monocytes into inflammatory (CDI Ib ⁺ CCR2 ⁺ GRI ⁺ CD62L ⁺ CX ₃ CRi ^low ) and non-inflammatory (CDI Ib ⁺ CCR2 ^" GRI ^" CD62L ^" CX ₃ CRi ^high ) monocytes (Geissmann F, Jung S, Littman DR (2003).) Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity, Vol. 19 (1), pp. 71-82.) In humans, these are Both monocyte populations have been known for some time and are mainly determined by the expression levels of the surface markers CD 14 and CD 16 (Ziegler-Heitbrock HW (2000) Definition of human blood monocytes J Leukoc Biol., Vol. 67 (5), S 603-6 .).

In a further embodiment, for the treatment in addition to the antibody against CCR2 additionally at least one antibody is used which is specific for monocytes, preferably selected from the group consisting of CD 14, CD33, CD64, CD68, CD91, CD15, and CD163. Particularly preferred is CD14. Such an embodiment may be particularly advantageous when the subject is a primate, in particular a human.

In another embodiment, the present invention relates to a method of depleting monocytes comprising contacting the monocytes with an antibody capable of specifically binding to the chemokine receptor CCR2. This may include procedures that represent a surgical or therapeutic treatment of the human or animal body. Preferably, the contacting takes place in vitro. The term "in vitro" is understood in this context in its broadest possible form. He refers to any process that takes place outside of the living body, so in particular to methods in cell culture, tissue culture or organ culture. In particular, the term in vitro is also understood to mean a process which relates to the treatment of blood outside the body of the subject.

The present invention further relates to a method of treating multiple sclerosis and / or rheumatoid arthritis in a subject, comprising the step of administering an antibody capable of specifically binding to the chemokine receptor CCR2. The present invention further relates to a method of depleting specific monocytes in a subject, comprising the step of administering an antibody capable of specifically binding to the chemokine receptor CCR2.

All of the preferred embodiments and variants of the invention and definitions already described above naturally also relate to the two aforementioned methods in a corresponding manner.

DESCRIPTION OF THE FIGURES: FIG. 1

Kinetics of monocyte depletion. A daily injection of 10 μg of MC-21 or isotype control antibody resulted in the depletion of GR1 + peripheral blood monocytes from mice for at least 5 days. We examined 5 mice per group. The blood samples were taken about 7 hours after the antibody injections. On the ratio of GRI + monocytes to CDl 9+ B cells (which are not significantly affected by the Ak treatment), the depletion of GR1 + monocytes persisting for at least 5 days is detected by the MC-21 antibody, day, day

Fig. 2

MC-21 induced high levels of IL-6 release. C57BL / 6 mice were treated intraperitoneally with 100 μg or 10 μg of MC-21 antibody. 2 hours after the injection, IL-6 levels in the plasma were measured by ELISA (enzyme-linked immunosorbent assay). Only in high dose did the MC-21 antibody lead to the release of IL-6. anti-CCR2, MC-21 antibody; i.p., intraperitoneally

Fig. 3

A. Different MC-21 concentrations differentially modulated the course of arthritis. In the model of collagen-induced arthritis (immunization of DBA / 1 mice with 100 μg of collagen on day 1 in complete Freund's adjuvant and on day 21 with Freund's incomplete adjuvant) daily administration (day 21-32) of high-dose MC-21 resulted Antibody (50 μg) significantly worsened arthritis, while daily administration of low-dose MC-21 (10 μg) resulted in a marked improvement in arthritis. B. The daily low-dose administration of the MC-21 antibody (anti-CCR2) (10-20 μg / day ip) was therapeutically effective in the collagen-induced arthritis model even when the arthritis was already significantly advanced. The antibody MC-67 was used as an isotype control in the same dosage. The mice were immunized 2 times 3 weeks apart with collagen (200 μg in complete and incomplete Freund's adjuvant). Only after the appearance of clear arthritis signs (score 3) was the antibody treatment started (day 0). The administration of MC-21 stopped the disease, while in the control group (administration of MC-67) the disease progresses significantly. The difference between the MC-21 and MC-67 groups was significant (p <0.05 for days 1-6). The increase in arthritis from day 0 was highly significant in the MC-67 group. In the MC group, the change in arthritis was not significant.

FIG. 4 The depletion of inflammatory monocytes improved the course of experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis. The application of 20 μg MC-21, but not of its isotype control (IgG2b), significantly improved the course of the disease. The MC-21 antibody or isotype control was given daily from day 17 (peak of disease) until day 34 after immunization. A clinical severity score of 2.5 corresponds to a significant hind leg weakness with unilateral hindlimb paralysis. A score of 1.0 corresponds to tail paralysis with preserved hindleg strength / motor function, daily, daily; clinicial EAE score, clinical EAE score; days after immunization, days after immunization

Fig. 5

Lower myelin damage during EAE in MC-21 treated mice 35 days after immunization. Quantification of demyelination. MC-21 treated animals had significantly more preserved myelin. IgG2b, isotype control; demyelination, demyelination

Fig. 6

The injection of antibodies DOC-2 and DOC-3 resulted in contrast to the injection of a

Isotype control antibody (5 mg / kg body weight) to a reduction in the number of CCR2-positive monocytes in the peripheral blood of marmoset monkeys. Shown is the relative proportion of CCR2-positive monocytes in the total number of leukocytes, this value being normalized to 100% in the group treated with isotype-control antibodies for the respective days. CCR2 ⁺ peripheral blood monocytes, CCR2 positive peripheral blood monocytes; iηjected antibody at 0 h, injected antibody at 0 h; Isotype Ko, isotype control

Fig. 7

The depletion of inflammatory monocytes in non-human primates leads to a stabilization of the weight (A.) as well as to a reduction of the neurological deficits (B.). Adult female marmosets (Marmoset, Callithrix jacchus) were treated with the anti-human CCR2 antibody DOC-2 and with the isotype antibody IgG1 (5 mg / kg i.p., respectively). The application of the antibodies was started as soon as the animals showed the first clear neurological symptoms. The marmosets were subsequently treated every two days.

Fig. 8

Therapeutic administration of the anti-human CCR2 antibody results in a significantly increased lifetime when treatment is initiated at the time of disease onset. Adult female marmosets (Marmoset, Callithrix jacchus) were incubated with the anti-human CCR2 antibody DOC-2 (anti-CCR2, 5 mg / kg ip, n = 2) or PBS (n = 2) from the onset of the disease and subsequently all treated for two days to death.

In the following embodiment, the invention is explained in more detail, without this being to be understood as a limitation of the scope of the invention.

Embodiment:

The MC-21 antibody as a means of depleting inflammatory monocytes in vivo: Monoclonal antibody MC-21 (Mack M, Cihak J, Simonis C, et al .. Expression and characterization of chemokine receptors CCR2 and CCR5 in mice, J Immunol. 2001 vol 166 (7), pp. 4697-704) binds specifically to murine CCR2 and shows none Cross-reaction with the closely related murine CCR5 receptor. He showed a strong binding to monocytes, here the subpopulation of GRl + monocytes in the mouse is recognized by the antibody.

Effect and kinetics of the MC-21 antibody in vivo: A single intraperitoneal injection of the MC-21 antibody into C57BL / 6 mice resulted in a virtually complete depletion of the inflammatory subpopulation of the monocytes (CCR2 + GR1 + monocytes) from blood and spleen (FIG. , Depending on the injected dose of MC-21, the depletion of the monocytes persisted for different lengths of time. At an injection of 5-10 μg, we observed a complete depletion of the GR1 + monocytes after 24 hours, while at 48 hours an occurrence of GR1 + monocytes was again observed. Injecting a higher dose of MC-21 (100 or 500 μg) resulted in a 2-3-day monocyte depletion (corresponding to the longer presence of the antibody in the plasma). Repeated injections of MC-21 antibody allowed depletion of monocytes to be sustained for approximately one week (Figure 1). Thereafter, the onset of inhibitory mouse anti-rat antibodies resulted in a loss of CCR2 binding ability of MC-21 antibodies. The MC-21 mediated depletion of GR1 + monocytes was largely mediated by Fc receptor since co-injection of antibodies to the Fc receptors FcgRIII and FcgRII by means of anti-CD16 / 32 (clone 2.4G2 ATCC HB197), MC-21 mediated depletion in Blood and spleen reduced. It is believed that the non-CD16 / 32 antibody-inhibitable portion of MC-21 mediated monocyte depletion is due to a complement-dependent mechanism or CD64-based ADCC mechanism.

The MC-21 antibody also had effects on CCR2-expressing basophilic granulocytes. Incubation of complete murine bone marrow with the MC-21 antibody resulted in a significant release of IL-6. This IL-6 release was dependent on basophilic granulocytes, since the depletion of the basophilic granulocytes from the complete bone marrow led to a significantly reduced MC-21 induced IL-6 release. Isolated basophilic granulocytes (isolation by means of magnetic beads against DX-5 and subsequent FACS sorting by means of CD45 and DX-5 expression) were also stimulated by incubation with MC-21 antibodies to release IL-6. Basophilic granulocytes from CCR2 deficient mice were not stimulated to release IL-6 by MC-21. These data indicate that MC-21 induced activation of basophilic granulocytes and that this activation occurred by binding of MC-21 to CCR2. As basophilic granulocytes also show strong expression of Immunoglobulin Fc receptors showed (especially CD 16/32), we assume that these receptors are important for the immobilization of MC-21 and may also contribute to the basophil activation. In vivo, basophil granulocyte activation was dependent on the dose of MC-21 administered. Upon injection of 10 μg of MC-21, we did not observe IL-6 release in vivo, whereas markedly induced IL-6 release upon injection of 100-500 μg (Figure 2).

Since monocyte depletion was already achieved with low doses of MC-21 (eg 5-10 μg) and IL-6 had unfavorable effects on the development of arthritis, repeated injection of MC-21 at low doses resulted in the possibility of monocyte depletion without causing simultaneous basophil activation by MC-21.

The study used the model of collagen-induced arthritis (CIA) in male DBA / 1 mice, a model that comes very close to human rheumatoid arthritis (RA) in many respects, and to the preclinical development of important therapies currently used in the clinic is used. In the model of collagen-induced arthritis, arthritis is induced by single (day 1) or two (day 1 and day 21) immunizations with mostly bovine collagen.

Treatment with antibodies directed against CCR2 from day 1 of the disease model interfered with collagen immunization and, therefore, joint inflammation was lower.

In contrast to the inhibitory effect of anti-CCR2 antibodies at the time of primary immunization with collagen, the application of the blocking CCR2 antibody MC-21 at elevated concentration (500 μg / mouse every 3 days) at a later time (from day 4, day 9, or day 21) a significant worsening of clinical and histological severity. This effect was associated with an increased humoral immune response to collagen (significantly higher anti-collagen antibody titers). CCR2 deficient mice backcrossed to the CIA susceptible strain DBA / 1 also showed increased arthritis (Quinones MP, Ahuja SK, Jimenez F, Schaefer J, Garavito E, Rao A, Chenaux G, Reddick RL, Kuziel WA, Ahuja SS (2004) Experimental arthritis in CC chemokine receptor 2-null mice closely mimics severe human rheumatoid arthritis J Clin Invest., Vol. 113 (6), pp. 856-66.). These data suggested that inhibition of CCR2 following induction of arthritis or absence of CCR2 significantly increased the rate of arthritis throughout the course of the disease. Therefore, according to the literature, CCR2 was considered to be an unsuitable target for the treatment of inflammatory rheumatic diseases.

Surprisingly, it could now be shown within the scope of the invention (FIG. 3 A) that treatment with lower-dose MC-21 antibodies (here 10 μg intraperitoneally per day) also led to a marked improvement in the arthritis during the progression phase of collagen-induced arthritis , MC-21 antibodies were given only from day 21 onwards. In comparison, day 21 treatment with a higher dose of MC-21 (50 μg / day) resulted in a significant worsening of arthritis. Also, a dose of antibodies against IgE (from day 21), which led to an activation of basophilic granulocytes, caused a significant deterioration of the arthritis.

Monocyte Depletion in the MS Model: In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), the disease could be induced by various myelin components (eg MOG _35- S ₅ ) or by adoptive transfer of autoreactive T cells to these components.

In another work, a selective small molecule CCR2 antagonist was shown to positively affect the course of EAE when given prior to the onset of more severe disease symptoms (Brodmerkel CM, Huber R, Covington M, Diamond S, Hall L, Collins R ₅ Leffet L, Gallagher K, Feldman P, Collier P, Stow M ₅ Gu X ₅ Baribaud F, Shin N, Thomas B ₅ Burn T, Hollis G, Yeleswaram S, Solomon K, Friedman S, Wang A, Xue CB, Newton RC ₅ Scherle P ₅ Vaddi K (2005) Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344, J Immunol., Vol. 175 (8), pp. 5370-8. / The underlying mechanism could be found in Brodmerkel and However, it may be questioned whether inhibition of monocyte migration into the brain is still therapeutically effective if a large number of monocytes are already present in the brain at a later stage of the disease The results of Brodmerkel are based on influencing the artificial release of the disease in the model system. Surprisingly, it has now been shown within the scope of the invention that a treatment regime based on CCR2 is effective as a therapeutic in the EAE stage of the progression or effector phase, ie during the disease. 20 μg of the monoclonal anti-CCR2 antibody MC-21 or the corresponding isotype control (IgG2b) were administered intraperitoneally daily from day 17 (peak of the disease) until day 34 after primary immunization, ie starting from the peak of the disease (EAE score at 2, 5, ie hind limb weakness with unilateral paralysis of the legs). A clear and statistically significant reduction of the course of the disease could be observed (FIG. 4). Clinical severity 1.0 means a tail paralysis. Interestingly, in contrast to the arthritis model, a comparatively high dose of MC-21Abs (250 μg) led to an improvement of the clinical course. A functionally relevant basophil activation in the EAE model by MC-21 could be detected neither by an increase of IL-6 in the serum of treated animals nor by a modulation of the disease course by anti-IgE administration.

The very significant and statistically significant improvement in clinical severity was accompanied by a significant reduction in infiltrating mononuclear cells in the central nervous system, as exemplified by the decreased number of CD3 ⁺ T cells and M AC-3 ⁺ macrophages in MC-21 treated animals at day 35 could be shown after immunization.

Likewise, the tissue damage was significantly lower in treated animals. For example, the degree of demyelination was significantly less pronounced in MC-21 treated animals than in the isotype control group.

To determine which cytokines and chemokines are formed locally in the CNS in MC-21 treated and thus monocyte-depleted animals, a quantitative PCR was performed. The spinal cord of treated animals was taken on day 35 after immunization, the RNA extracted, reverse transcribed and quantitated by a quantitative real time (RT) PCR. It found a much lower

Production of chemoattractive and toxic proteins, e.g. IP-10, MCP-I and MIP-Ia in the CNS of MC-21 treated animals.

The next step was to investigate whether primates (White-bellied monkeys, Callithrix jacchus) cross-react with antibodies to human CCR2. As antibodies against human CCR2 we have thereby analyzed the antibodies produced in the invention DOC-I, DOC-2 and DOC-3. To prepare the DOC antibodies, at least 6 weeks old BALB / c mice were intraperitoneally immunized 6-8 times at least 3 weeks apart with at least 10 million CHO cells stably transfected with full-length CCR2b. Four days after the last immunization, spleen cells were fused with X63.Ag8.653 myeloma cells and cultured in selection medium. The supernatant of the resulting hybridomas was tested by FACS analysis for binding to CCR2b transfected or as a control for binding to CXCR4 transfected CHO cells. After duplicate recloning of positive cultures several monoclonal antibodies (DOC-I, DOC-2 and DOC-3) were obtained which specifically bind to human CCR2 and did not cross-react with murine CCR2 or CCR5, as well as with human CCR1, CCR3, CCR5 or CXCR4 have. The DOC antibodies bound to a subpopulation of T cells (approximately 20-30%) in human peripheral blood, as well as to the majority of CD 14+ monocytes (> 95%). By preincubating the human cells with MCP-I (1 μg / ml) for 30 min at 37 ° C, the binding of CCR2 antibodies to the above-described leukocyte subpopulations was virtually completely prevented, resulting in MCP-I-induced internalization of CCR2 explain is. This additionally showed the specificity of the DOC antibodies.

The DOC antibodies have been extensively characterized in terms of their functional properties. A summary is shown below:

Docl: possesses the isotype IgG3 binds to Loopl, 2 and 3 of the extracellular CCR2 domains do not induce CCR2-down modulation on human peripheral blood monocytes (PBMC) at 37 ° C, blocking MCP-I-induced CCR2-down modulation human PBMC blocks MCP-1 and MCP-3 induced chemotaxis of Monomacό cells and human PBMC shows partial inhibition of MCP-I binding and MCP-I induced

Calcium influx

Doc2: possesses the isotype IgGl binds to Loopl + first half of loop2 of the extracellular CCR2 domains induced a strong CCR2-down modulation on human PBMC at 37 ° C, blocking the MCP-I and MCP-3 induced chemotaxis of Monomacό cells and human PBMC showing a very effective inhibition of MCP-I binding and of MCP-I induced concentration calcium influx

Doc3: possesses the isotype IgGl binds to Loopl + second half of loop2 of the extracellular CCR2 domains induced a weak CCR2 down modulation on human dependent concentration

PBMC blocks the MCP-I and MCP-3 induced chemotaxis of Monomacό cells and human PBMC showing a very effective inhibition of MCP-I binding and MCP-I induced

Calcium influx

The chemotaxis was measured in this context using Transwell filter inserts (3 μm pore size for PBMC and 5 μm pore size for Monomac®), with the cells (PBMC or Monomac 6) in the upper well and the chemokines (20-200 ng / ml ) were placed in the lower well. After incubation for approx. 60 min. the number of cells migrated into the lower well was measured.

Calcium influx was measured in this regard by an assay based on anequorin and luminescence (see Blanpain et al, Molecular Biology of the Cell, Vol. 13, 723-737, 2002). DOC antibody binding to CCR2 was measured by flow cytometry in which a dye-labeled rabbit anti-mouse Ig antibody was used as the secondary antibody.

To determine the binding epitopes of the antibodies, binding to cells expressing chimeric receptors consisting of CCR2 and CCR5 was determined. The internalization (down-modulation) of CCR2 was also measured by flow cytometry.

The cross-reaction of antibodies DOC-I, DOC-2 and DOC-3 with CCR2 on white tufted leukocytes {Callithrix jacchus) was investigated by FACS analysis. For this purpose, whole blood of marmoset monkeys was incubated with 5 μg / ml of the CCR2 antibodies or with the same concentration of isotype control antibodies (IgG1 and IgG3), followed by a PE (phycoerythrin) -labeled secondary antibody (rabbit (F (ab) 2 fragment anti-mouse Ig, R0439, DakoCytomation) After erythrocyte lysis, FACS analysis was performed to determine the specificity of DOC antibody binding In addition to white marmoset CCR2, internalization of CCR2 with human MCP-I was induced in part of the whole blood before incubation with the DOC antibodies, whereas whole blood was incubated with 1 μg / ml MCP-I at 37 ° for 30 min C was pre-incubated and then tested for DOC antibody binding as described above, showing strong binding of antibodies DOC-2 and DOC-3 and a slightly lower binding of DOC-I antibody to the forward-sideward scatter of the FACS dot About 50% of the monocytes thus bound bound DOC-2, DOC-3, and DOC-I, and this binding was completely lost when pre-incubated leucocytes were used in MCP-1 Bodies showed no binding to monocytes. Apart from the monocyte subpopulation, binding of the DOC antibodies was only found on a very small additional leukocyte subpopulation, which can be classified as basophilic granulocytes due to their forward-sideward scatter. The monocyte population recognized by the DOC antibodies was homogeneous CDI Ib positive, as demonstrated by staining with the antibody clone (M 1/70, rat-anti mouse CDI Ib). This CDI Ib antibody showed a cross-reaction with human and also marmoset CDI Ib.

In addition, the pharmacokinetic properties of DOC antibodies were investigated. At 24, 48 and 96 hours after injection of antibodies DOC-2 and DOC-3 (5 mg / kg body weight), the plasma concentrations of the antibodies were measured. For this purpose, monkey plasma was incubated in different dilutions with hCCR2b-transfected CHO cells and the plasma concentration of the DOC antibodies was calculated on the basis of a standard curve consisting of different dilutions of the antibodies DOC-2 and DOC-3:

In addition, it was investigated whether there was a depletion of the CCR2 positive monocytes after injection of the DOC-2 or DOC-3 antibodies. For it was after 24h, 48h and 96h Injection of the antibodies DOC-2 and DOC-3 or an isotype control antibody (5 mg / kg body weight, each 2 monkeys per group) blood and decreased the proportion of CCR2 positive monocytes in the total number of leukocytes by FACS analysis certainly. Whole blood was, as described above, stained with the antibodies DOC-2 and DOC-3 followed by a phycoerythrin-labeled secondary antibody and identified in conjunction with the forward-sideward scatter the CCR2 positive monocytes. Figure 7 shows that the proportion of CCR2 positive monocytes in the total number of leucocytes in the DOC-2 or DOC-3 treated animals was reduced (to about 50%) on the first day after injection. The proportion of CCR2 positive monocytes in the total number of leukocytes in the isotype control antibody-treated group was set equal to 100% at the respective days.

After demonstrating the efficacy of anti-human DOC-2 AK with high-grade depletion of inflammatory monocytes in the peripheral blood of marmoset monkeys, we sought to investigate the efficacy of this depleting AK in primate EAE disease models. For this, adult (about 3 years old) female marmosets (Marmoset, Callithrix jacchus) were immunized with the recombinant rat MOG protein in complete Freund's adjuvant (KFA) (50 μg MOG in 600 μl KFA). After 3-4 weeks, the animals began to show first neurological symptoms and inappetence. We began the therapeutic application with the anti-human CCR2 antibody DOC-2 and the corresponding isotype control (IgGl) in the first clear neurological disease symptoms. The marmosets were then treated every two days with DOC-2 AK or isotype AK (5 mg / kg i.p.). The DOC-2 AK-treated primates showed weight stabilization and a decrease in neurological deficits during therapy, as opposed to control. In another experiment, which started with the application of the antibodies at the time of the disease, we were able to demonstrate a significantly longer survival time for the lethal EAE in comparison to the control animals.

In summary, the data determined in the context of the invention show for the first time that an antibody which dejected CCR2 +, GR1 + inflammatory monocytes in vivo had a clear therapeutic effect both in the mouse model and primate model of MS and in the mouse model of arthritis. FACS analysis: EDTA anticoagulated whole blood or splenocytes were incubated on ice with directly labeled or unlabeled antibodies for 45 min and then removed by washing 3 times. As directly labeled antibodies (labeling with the colorants FITC, PE, PE-Cy5, APC) against murine cells were used: antibodies against CDI Ib, GRI, CD4, CD19, CD45, DX5, IgE (all from BD-Pahrmingen). Monocytes were identified by their forward and sideward light scatters and by their expression of CDI Ib and divided into GrI + and GRI monocytes after expression of GrI.

The non-directly labeled antibodies used were the CCR2 antibody MC-21 (5 μg / ml) and a rat IgG2b isotype control antibody (5 μg / ml, BD-Pharmingen), followed by a biotinylated mouse anti-rat antibody. IgG2b (5 μg / ml, BD-Pharmingen) followed by 10% rat serum, followed by streptavidin-PE or streptavidin-PE-Cy5 (DakoCytomation) and optionally other directly labeled antibodies. The unlabeled antibodies DOC-1, DOC-2 and DOC-3 and the corresponding isotype controls (Sigma-Aldrich) were followed by a PE-labeled polyclonal rabbit anti-mouse F (ab) 2 fragment (DakoCytomation, R0439).

After staining, erythrocyte lysis with FACS-Lysing Solution (BD-Pharmingen) took place. The FACS analysis was performed on a FacsCalibur device from BD-Pharmingen.

ELISA: The measurement of IL-6 in the culture supernatant or EDTA plasma was carried out with a commercial ELISA (OptEIA) from BD-Pharmingen.

Cell Isolation / Cell Depletion: Naïve basophils were isolated by enrichment of DX-5 positive cells from spleen and bone marrow of mice using DX-5 magnetic beads (Miltenyi, isolation according to instructions). DX-5-APC and CD45-FITC were subsequently stained to isolate the basophilic granulocytes (DX5 +, CD45 low) using FACS sort (BD, FACS ARIA). The purity of the basophilic granulocytes thus obtained was over 95%.

For the depletion of basophilic granulocytes, the starting cells were incubated with anti-IgE-FITC antibodies, washed and then incubated with anti-FITC beads and depleted by magnetic sorting (Miltenyi). The depletion was over 95%. Collagen-Induced Arthritis: At least 8 week old male DBA / 1 mice were immunized on day 1 with 100 μg bovine collagen (Sigma, Cl 188) in complete Freund's adjuvant and on day 21 with 100 μg collagen in incomplete Freund's adjuvant subcutaneously at the tail base , The clinical arthritis score was determined by taking a score for each paw and adding it up for all four paws: score per paw: 1 for joint swelling, 2 for at least 2 joint swelling, 3 for total paw swelling, 4 at deformation of the paw. Antibody treatment was as described in the illustrations. - ^■

Experimental autoimmune encephalomyelitis (EAE) in the C57BL / 6 mouse: For active immunization 6-8 weeks old mice were injected subcutaneously with 200 ug of the MOG ₃₅ _ ₅₅ peptide, emulsified in CFA (complete Freund's adjuvant) (supplemented with 1 mg / ml from Mycobacterium tuberculosis (Difco)), injected; followed by 250 ng of pertussis toxin iv on the same day and 2 days later. The clinical manifestation of EAE usually began between the 13th and 18th day after immunization. The mice were then neurologically estimated 4 times a week for the following criteria: 0, no visible neurological symptoms; 0.5, flaccidity of the distal tail; 1, flaccidity of the entire tail; 1.5; Slackness of the entire tail and weakness of the hind legs; 2, unilateral partial paresis of the hind legs; 2.5, bilateral partial paresis of the hind legs; 3, complete paresis of the hind legs; 3.5 complete paresis of the hind legs and unilateral paresis of the front legs; 4, complete paresis of all 4 extremities; 5, dead.

Recombinant rat MOG was emulsified in Complete Freund's Adjuvant (KFA) in EAE in White-marmoset (Callithrix jacchus) (50 μg MOG in 600 μl KFA). In 150 μl portions, the emulsion was applied subcutaneously to the back at four sites. Two application sites were in the shoulder and the other two in the pelvic area. Before the application, the corresponding parts of the body were shaved and the skin disinfected.

Instead of M. tuberculosis, we used KFA Mycobacterium buytricum at a reduced dose of 1 mg / ml KFA. These modifications usually made it strong effective KFA mitigated without, however, losing its effect. This immune stimulation was necessary to trigger the autoimmune reaction.

Real-Time PCR (RT-PCR): For this purpose, the animals were killed, the spinal cord freshly taken and rinsed briefly with sterile PBS. The total RNA was isolated by Trizol reagent according to the manufacturer's instructions rewritten in cDNA and carried out the RT-PCR.

Histology: The mice were killed by CO ₂ . The spine was removed and placed in 10% buffered formalin. After fixation, the spinal cord was removed, embedded and either with H & E for assessment of

Leukocyte infiltration or with Luxol fast blue (LFB) to visualize the myelinated

Dyed fibers. Other immunohistochemical stains concluded different

Blood cell types (MAC-3 for macrophages: BD Pharmingen; CD3 for T cells: Serotec, Dusseldorf).

Claims

claims

A medicament containing an antibody capable of specifically binding to the chemokine receptor CCR2 for the treatment of multiple sclerosis and / or rheumatoid arthritis in a subject.

A medicament according to claim 1, wherein the subject is a primate, preferably a human.

3. Medicament according to one of claims 1 to 2, for the treatment of therapy refractory multiple sclerosis and / or multiple sclerosis in an advanced stage.

A medicament according to any one of claims 1 to 2 for the treatment of therapy-refractory rheumatoid arthritis and / or rheumatoid arthritis at an advanced stage.

The medicament according to any one of claims 1 to 4, wherein the antibody is selected from the group consisting of Doc-1, Doc-2, Doc-3, and MC-21.

A medicament according to any one of claims 1 to 5 wherein the treatment is with an effective dose of the medicament chosen so as not to exceed that dose necessary to achieve a 95%, preferably 90%, depletion, more preferably To reach 80% of CCR2-expressing monocytes in peripheral blood.

A medicament according to claim 6 wherein the treatment is with an effective dose of the medicament chosen to deplete at least 20%, preferably at least 30%, of the CCR2-expressing monocytes in the peripheral blood.

A medicament according to any one of claims 1 to 5 wherein the treatment is with an effective dose of the medicament chosen to be no more than a 20-fold increase, preferably not more than a 10-fold increase, in the level of interleukin-6 (IL-6) and / or interleukin-4 (IL-4) in the blood plasma.

A medicament according to any one of claims 1 to 5 wherein the treatment is with an effective dose of the medicament chosen to correspond to 0.01-20 mg of the antibody per kg body weight of the subject per day.

A medicament containing an antibody capable of depleting monocytes for the treatment of multiple sclerosis in a subject.

A medicament according to claim 10, wherein the medicament is further defined as in one or more of claims 2 to 3 and 5 to 9.

A medicament containing an antibody capable of depleting monocytes for the treatment of rheumatoid arthritis in a subject.

The medicament according to claim 12, wherein the medicament is further defined as in one or more of claims 2 and 4 to 9.

14. A method for depleting monocytes, comprising contacting the monocytes with an antibody that is capable of binding to the chemokine receptor CCR2, in particular excluding methods involving a surgical or therapeutic

Represent treatment of the human or animal body.

A method of treating multiple sclerosis and / or rheumatoid arthritis in a subject, comprising the step of administering an antibody capable of specifically binding to the chemokine receptor CCR2.

16. The method according to claim 15, wherein the subject is a primate, preferably a human.

17. The method according to any of claims 15 to 16, wherein the multiple sclerosis is a therapy-refractory multiple sclerosis and / or multiple sclerosis in an advanced stage.

A method according to any one of claims 15 to 16, wherein the rheumatoid arthritis is a therapy refractory rheumatoid arthritis and / or an advanced stage rheumatoid arthritis.

19. The method according to any one of claims 15 to 18, wherein the antibody is selected from the group consisting of Doc-1, Doc-2, Doc-3, and MC-21.

20. A method according to any one of claims 15 to 19, wherein the treatment is carried out at an effective dose chosen so as not to exceed that dose necessary to achieve a depletion of 95%, preferably 90%, more preferably 80%. to reach the monocytes in the peripheral blood.

The method of claim 20, wherein the treatment is at an effective dose selected to deplete at least 20%, preferably at least 30%, of the monocytes in the peripheral blood.

22. The method according to any one of claims 15 to 19, wherein the treatment is carried out at an effective dose, which is chosen so that it does not increase more than 20 times, preferably not more than a lOX, the level of interleukin-6 (IL -6) and / or interleukin-4 (IL-4) in the blood plasma.

A method according to any one of claims 15 to 19, wherein the treatment is at an effective dose selected to correspond to 0.01-20 mg of the antibody per kg body weight of the subject per day.

24. A method for depleting monocytes in a subject, comprising the step of administering an antibody capable of specifically binding to the chemokine receptor CCR2.

25. The method of claim 24, wherein the method is treating multiple sclerosis and / or rheumatoid arthritis in the subject.

26. Use of an antibody capable of specifically binding to the chemokine receptor CCR2 for the manufacture of a medicament for the treatment of multiple

Sclerosis and / or rheumatoid arthritis in a subject, wherein the medicament is preferably as defined in any one of claims 1 to 9.

27. Use of an antibody capable of depleting monocytes for the manufacture of a medicament for the treatment of multiple sclerosis and / or rheumatoid

Arthritis in a subject, wherein the medicament is preferably as defined in any one of claims 10 to 13.